(a) Field of the Invention
The present invention relates to a bio-adhesive derived from mussel, more particularly to a novel Mytilus galloprovincialis foot protein-3A (MGFP-3A) and a recombinant protein that is a hybrid of MGFP-3A, FP (Foot Protein)-1 and MGFP-5 (Mytilus galloprovincialis foot protein-5).
(b) Background of the Invention
Mussels produce and secrete specialized water-resistant bioadhesives, and have been studied as a potential source of water-resistant bioadhesives. They adhere tightly to surfaces underwater using the byssus secreted from the foot of the mussel. At the end of each thread is an adhesive plaque containing a water-resistant glue that enables the plaque to anchor to wet solid surfaces [Waite, J. H., Biology Review. 58:209-231 (1983)]. This strong and water-insoluble adhesion has attracted interest for potential use in biotechnological applications.
In addition, mussel adhesive proteins can also be used as medical adhesives as they are non-toxic to the human body and do not impose immunogenicity [Dove et al., Journal of American Dental Association. 112:879 (1986)]. Moreover, their biodegradable properties make them environmentally friendly.
The byssus can be divided into distal and proximal parts. The proximal part is connected to the stem gland of the mussel foot, while the distal part is connected to the adhesive plaques. The adhesive plaque is composed of five distinct types of proteins: foot protein type 1 (FP-1) to type 5 (FP-5) [Deming, T. J., Current Opinion in Chemical Biology. 3:100-105 (1999)].
All of the mussel adhesive proteins contain high ratios of 3,4-dihydroxyphenyl-L-alanine (DOPA), which is derived from hydroxylation of tyrosine residues [Waite, J. H., Biology Review. 58:209-231 (1983)]. The adhesive proteins closest to the adhesion interface have the highest proportion of DOPA residues [Waite, J. H., Integr. Comp. Biol. 42:1172-1180 (2002)]. In contrast, mussel adhesive protein analogs lacking DOPA show greatly reduced adhesion abilities [Yu et al., Journal of American Chemical Society. 121:5825-5826 (1999)]. Indeed, a biochemical study showed that DOPA residues can enable mussel adhesive protein molecules to cross-link with each other via oxidative conversion to DOPA o-quinone. Thus, the DOPA content of a mussel adhesive protein appears to be specifically related to its adhesive properties.
Currently Cell-Tak, a naturally extracted mussel adhesive protein product, is commercially available. This adhesive is mainly composed of FP-1 and FP-2 type proteins, with a minor portion of FP-3. However, the natural extraction process is labor-intensive and inefficient, requiring around 10,000 mussels for 1 g of protein [Morgan, D., The Scientist. 4:1-6 (1990)].
Therefore, researchers have sought to produce recombinant mussel adhesive proteins, for example FP-1, in expression systems such as Escherichia coli and yeast.
However, these previous studies failed to express functional and economical mussel adhesive proteins due to a number of complications, including a highly biased amino acid composition (5 amino acid types comprise ˜89% of the total amino acids in FP-1), different codon usage preferences between mussel and other expression systems (tRNA utilization problems) and low protein yields [U.S. Pat. No. 5,242,808, Filpula et al., Biotechnol. Prog. 6:171-177 (1990), Salerno et al., Applied Microbiology and Biotechnology 58:209-214 (1993), Kitamura et al., Journal of Polymer Science Part A: Polymer Chemistry, 37:729-736 (1999)].
The inventors of the present invention have separated MGFP-5 encoding a novel adhesion protein from a mussel and established a expression system for the production of an adhesion protein that is more adhesive than a FP-1 and is from a mussel (WO 05/02920) to overcome the problems in the prior art.
However, MGFP-5 has drawback of a low producing yield, and a necessity of improving solubility. And so, the inventors improve yield and solubility of polypeptide by developing the chimeric gene with FP-1 and MGFP-5 (WO 05/02920).
Therefore, an adhesive having excellent physicochemical properties including an adhesion force can be developed by combination with MGFP-5 and existing or novel polypeptide of a mussel.